Summary The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators.

Replication protein A (RPA) and RAD51 are DNA-binding proteins that help maintain genome stability during DNA replication. These proteins regulate nucleases, helicases, DNA translocases, and signaling proteins to control replication, repair, recombination, and the DNA damage response. Their different DNA-binding mechanisms, enzymatic activities, and binding partners provide unique functionalities that cooperate to ensure that the appropriate activities are deployed at the right time to overcome replication challenges. Here we review and discuss the latest discoveries of the mechanisms by which these proteins work to preserve genome stability, with a focus on their actions in fork reversal and fork protection.

SUMMARY RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis, but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.

Protein lysine methylation is a crucial post-translational modification that regulates the functions of both histone and non-histone proteins. Deregulation of the enzymes or'writers' of protein lysine methylation, lysine methyltransferases (KMTs), is implicated in the cause of many diseases, including cancer, mental health disorders and developmental disorders. Over the past decade, significant advances have been made in developing drugs to target KMTs that are involved in histone methylation and epigenetic regulation. The first of these inhibitors, tazemetostat, was recently approved for the treatment of epithelioid sarcoma and follicular lymphoma, and several more are in clinical and preclinical evaluation. Beyond chromatin, the many KMTs that regulate protein synthesis and other fundamental biological processes are emerging as promising new targets for drug development to treat diverse diseases.Covalent post-translational modifications (PTMs) of proteins are a major source of molecular functional diversity in mammalian cells, and their aberrant regulation is a ✉

RAD51 promotes homologous recombination repair (HR) of double-strand breaks and acts during DNA replication to facilitate fork reversal and protect nascent DNA strands from nuclease digestion. Several additional HR proteins regulate fork protection by promoting RAD51 filament formation. Here, we show that RADX modulates stalled fork protection by antagonizing RAD51. Consequently, silencing RADX restores fork protection in cells deficient for BRCA1, BRCA2, FANCA, FANCD2, or BOD1L. Inactivating RADX prevents both MRE11- and DNA2-dependent fork degradation. Furthermore, RADX overexpression causes fork degradation that is dependent on these nucleases and fork reversal. The amount of RAD51 determines the fate of stalled replication forks, with more RAD51 required for fork protection than fork reversal. Finally, we find that RADX effectively competes with RAD51 for binding to single-stranded DNA, supporting a model in which RADX buffers RAD51 to ensure the right amount of reversal and protection to maintain genome stability.